Human DNA (cytosine-5)-methyltransferases (C-5-MTase) are key regulators in the epigenetic control of gene expression through the S-adenosyl methionine(SAM)- dependent methylation of the DNA base cytosine. Hypermethylation of CpG islands occurs in nearly all cancers, often resulting in transcriptional silencing of tumor suppressor genes. Unlike mutations that promote cancer, such epigenetic events are reversible and amenable to pharmacological intervention, making human MTases attractive anticancer drug targets. We have fully developed the first robust, efficient and economical fluorescence assay suitable for in vitro high-throughput (HTP) screening of human DNA methyltransferase I (DNMT1). In this proposal we propose to use this assay to screen a custom library of MTase-directed compounds that will be constructed using our "substrate fragment tethering" (SFT) technology. Our future plan is to use this assay to rapidly evaluate various substrate fragment tethering strategies with the goal of discovering novel MTase inhibitors that will be useful for studying epigenetic regulation of gene expression or as leads for therapeutic development. PUBLIC HEALTH RELEVANCE: Hypermethylation of CpG dinucleotides in promoter regions of tumor suppressor genes by DNA cytosine-5-methyltransferase (MTase) enzymes is an important hallmark of human cancers. Such reversible methylation, known as epigenetic silencing, is a key pathway resulting in loss-of-function phenotypes that promote the growth of many cancers. Thus, inhibitors of MTase enzymes have the potential for reversing hypermethylation-induced gene silencing and are exciting new targets for epigenetic cancer therapies. This study describes new approaches to the discovery of such small molecule inhibitors.